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A review.Manufacturing recombinant adeno-associated virus (rAAV) is a complex process that hinges on various critical quality attributes (CQAs) to ensure the production of effective therapeutic products.The potency of rAAV products remains an elusive CQA, primarily due to a lack of representative bioassays and the innate complexity of how rAAV interacts within vivohost systems.A traditional indicator of potency is the presence of DNA cargo inside the viral capsid, specifically the quantity of filled capsids (with full transgene cargo), as opposed to the non-potent empty capsids (without cargo).In the study by Ebberink et al., the authors employed a novel combination of MP and nuclease treatment to probe structural integrity during the thermally induced uncoating process of rAAV particles.The study raises an important question about the factors driving the differences between rAAV particles of variable structural integrity.Integrating these relevant assays into realtime monitoring during the cultivation process may offer valuable insights into the production kinetics of particles that are more prone to enter a less "fragile" or intermediate uncoating stage.This information might correlate with higher stability and in vivo potency, potentially guiding process optimization for better therapeutic outcomes.

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